Effect Of Neurotrophin-3 Genetically Modified Olfactory Ensheathing Cells Transplantation On Spinal Cord Injury

Spinal cord injury (SCI) is very frequent in our society, the number of cases increasesevery year, and this pathologic condition often causes permanent and irreversible functionaldeficits in the patients. The devastating consequences of this affection have motivatednumerous scientists throughout the world to search in experimental animal models for a repairstrategy that could, in the future, be applied to humans. However, due to the limited neuronal survival, the lack of spontaneous axonalregeneration and the inhibitory glial environment, self-repair after damage of the mammaliancentral nervous system (CNS) is rather poor. Henceforth, the repair of brain and spinal cordinjury thus far is still a major therapeutic challenge. To be gratified, in recent years,substantial evidence has suggested that the neurons and the axons proximal to the site ofinjury were still alive after the impairment. Therefore, external interventions such as celltransplantation and neurotrophic factors delivery overcoming the inhibitory environment atthe injury site could stimulate some of the remaining intrinsic plasticity of neurons and, thus,to foster the healing of the otherwise nonregenerative CNS. At present, little of these strategies alone have been sufficient in repairment of adultCNS, two or more strategies used in combination might bring about adequate structural andfunctional recovery. During the past two decades, the dramatic progress in therapeutic allianceand in vivo and ex vivo gene therapy has heightened optimism about future cures for injury ofCNS. Among those cells candidate for transplantation, due to their ability both to support CNSaxon regeneration and to remyelinate the regenerated axons, olfactory ensheathing cells(OECs) have attracted more attention as potential therapeutic agents for the treatment of SCI.Research performed over the past decades demonstrated that the presence of OECs wouldcreate a more favourable and supportive environment for axonal growth, guide the axonsregenerate through the glia scar to CNS and promoted the recovery of sensorimotor functionsin paraplegic rats. Furthermore, the available evidence supports the view that OECs are more - 6 -第二军医大学博士学位论文likely to intergrate into the CNS microenvironment and migrate long distances within theneuropil of the CNS after grafting to repair the injured spinal cord. Thus OECs transplantationhas emerged as a very promising repair strategy in SCI. Neurotrophin-3 (NT-3), a member of neurotrophin super-family, are well known for theirbeneficial effects on survival of several neuronal cells, neuroprotection and neurite outgrowth.Local delivery of NT-3 can counteract pathological events and induce a regenerative responseafter SCI. Supplement of exogenetic NT-3 might be a new approach to SCI repair. In present experiment, we examined the effect of rat NT-3 gene modified OECstransplantation on SCI. Firstly, primary cultures of OECs were harvested from olfactory bulbof 2.5-month-old rat. Purification of OECs was developed with cytosine arabinoside (Ara-C)treatment and differential adhesion method. Then OECs were stimulated to propagate by BPEor forskolin. The purity of OECs was determined according to immunostaining positively forp75, S-100 and GFAP at different time. Viability and purity of OECs at distinct time wasobserved and compared. Meanwhile, TrkC mRNA and NT-3 mRNA of OECs and thepromoting effect of NT-3 to OECs survival was investigated. Secondly, adult SD ratsreceiving T9 spinal cord contusion were treated with NT-3 and/or OECs, and the ability totissue sparing, glia reaction, axon regeneration and neuron prevention was assessed. All ratswere behaviorally tested for 8 weeks using the Basso-Beattie-Bresnahan locomotor ratingscale and inclined plane test. Finally, Rat NT-3 gene was transfered into OECs usingretroviral system. The engineered OECs-N...